Cancer will continue to be an enormous global health problem unless markedly improved paradigms for combating this devastating disease are successfully implemented into clinical practice. Early detection of cancer is at the heart of timel treatments and potentially improved patient outcomes. At present no broadly applicable early cancer detection method exists as most technologies rely on detection of sparsely produced and non-specific endogenous cancer biomarkers. The long-term goal of this competitive renewal application is to develop an integrated, safe technology called tumor-activatable minicircles (MCs) that activate exogenous reporter gene(s) expression near-exclusively in tumor cells so that tumor(s) can be detected and localized in living subjects via both blood-based and molecular imaging assays. Advantages of this strategy include minimal/near-zero background signal in tumor-free individuals, temporal control of reporter gene expression, and a highly modular system that can be iteratively improved to maximize safety, sensitivity, and specificity. An additional goal is to develop a secretable RNA-based blood reporter that can be applied to many clinical applications. Excellent progress on the development and utilization of novel reporter gene technologies has been made over the last 5-year funding period. We have engineered optimal bioluminescence resonance energy transfer systems for imaging drug-modulated protein-protein interactions in deep tissues in small living subjects, and discovered novel small molecular inhibitors of critical interactions involved in tumor progression. Now we will combine advances in the development of potent and safe MC vectors with our continued efforts in translating reporter gene technologies from small animals to patients and propose that reporter-gene expressing tumor-activatable MCs can be used for cancer detection. Our overall goal is to innovate clinically-relevant safe reporter gene systems for expanding the clinical uses of reporter genes for early cancer detection and management. The specific aims of the current renewal are to optimize delivery of MCs to tumors in vivo (Aim 1), to develop dual-reporter MCs that can be used to detect cancer with both secretable blood-based and pre-clinical bioluminescence imaging assays (Aim 2), and to develop an optimal protein- or RNA-based blood reporter that can be used in conjunction with a clinically-relevant positron emission tomography (PET) reporter for cancer detection and localization (Aim 3) in living subjects. Through continued refinement of our highly modular strategy the significance of this work will be to provide a powerful cancer detection technology that may allow earlier such that more efficacious treatment paradigms can be implemented. We envision our system could first be utilized in cancer patients with high-risk of tumor recurrence, followed by screening high-risk populations prior to initial tumor diagnosis. Ultimately, our safe and optimized detection systems may serve as powerful screening tools that will reduce cancer-related morbidity and mortality.